Method for preserving and stabilizing proteins for formulations of sanitary, pharmaceutical and cosmetic products

ABSTRACT

A method for preserving and stabilizing proteins includes the steps of forming an anhydrous medium having oily phase components having hydrophilic residues, and dispersing the proteins into the anhydrous medium under ambient pressure and temperature conditions so that the proteins are incorporated into the anhydrous medium so as to maintain an active formation of the proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patent application Ser. No. 14/759,320, filed on Oct. 14, 2015 and entitled “Method for Preserving and Stabilizing Proteins, Which Can be Used for Industrial Development of Formulations of Sanitary, Pharmaceutical and Cosmetic Products”, presently pending.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not applicable.

INCORPORATION-BY-REFERENCE OF MATERIALS SUBMITTED ON A COMPACT DISC

Not applicable.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a method for preserving and stabilizing proteins. This method of preserving and stabilizing proteins relates to the industrial development of formulations of sanitary, pharmaceutical and cosmetic products. Additionally, the present the present invention relates to the industrial chemical sector dedicated to the manufacture of pharmaceutical and cosmetic products, and in particular those intended to preserve growth factors.

2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.

Currently the stability in proteins has attracted considerable interest in the chemical, pharmaceutical and cosmetic field. Proteins are used as active substances of numerous treatments in diseases such as diabetes, cancer, haemophilia, myocardial infarction, just to cite some pathologies [Krishnamurthy and Manning, 2002]. Note that the use of protein structures such as growth factors, e.g. the EGF, aroused a great interest in the cosmetic industry in recent years. So, if a protein can not be properly stabilized, it may lose its native structure with consequent loss of its biological activity [Krishnamurthy and Manning, 2002].

The problem is that the protein stabilization is particularly difficult since they are very susceptible to degradation phenomena: physical, chemical and enzymatic. Chemical degradation is related to deamination, oxidation, reduction, hydrolysis processes and chemical interactions such as disulfide bond interactions. Physical degradation includes surface adsorption, aggregation, dissociation, denaturation, and photolysis processes. In addition, there are factors that influence the aggregation of proteins related to the properties of the dispersion medium, such as temperature (related to the thermodynamics and kinetics of transformation of the structural protein conformation), pH (related to interactions of positive or negative charges with the protein residues), ionic strength (related to salts and their concentration to interact with charged groups) and surfactants (related to conformational thermodynamic stability) [Chi et al., 2003].

There are technological processes to ensure that proteins remain for longer period of time with its native conformation, said processes can be carried out by physical processes such as freezing (below −10° C.) or lyophilisation (for the elimination of the humidity present in an aqueous solution of protein), however, even the products obtained by these methods suffer from degradation; or chemical processes through the addition of co-solvents may be carried out [Chang and Pikal, 2009].

The EGF was the first polypeptide isolated and characterized as a growth factor. It has a biological activity related to its native structure capable of stimulating the proliferation of keratinocytes and fibroblasts (with the consequent formation of collagen), induces angiogenesis (formation of new vessels) and performs subsequent vascularisation of the area where it is applied. These properties promote the appearance of new skin with a considerable thick, restoring its elasticity and firmness, thus diminishing the unwanted effects of cellular oxidation and therefore resulting in the elimination of wrinkles [Tang et al., 1994]. Such growth factor has begun to use recently in topical formulations, where very good results have been obtained related to tissue regeneration, the acceleration in the healing of burns, treatment of keloid, acne and stretch marks, even improving outcomes of treatments of surgical type, promotion of the consolidation of skin grafts as well as the post-peeling application. However, said proteins are increasingly used in the pharmaceutical and cosmetic industry, but have not been used massively due to its high prices and their difficult stabilisation [Schouest et al., 2012].

Fibroblast growth factor (bFGF) is a growth factor that acts to increase the mitotic activity index and DNA synthesis, facilitating the proliferation of various precursor cells, such as chondroblasts, collagenoblasts, and osteoblasts, etc., that form the body's fibrous, connective, and support tissues. It contributes to wound healing, haematopoiesis, angiogenesis, or the embryonic development. To this end, they perform very different functions: a) they contribute to the re-epithelialisation of the tissues damaged during healing; b) they have blood vessel formation inducing-activity; c) they are involved in processes for differentiation of the blood cell lines; and, d) they are involved in the differentiation of skeletal and cardiac muscle, the maturation of the lungs and the specification of the hepatocytes from endoderm cells.

The object of the present invention is, therefore, the development of a new method for preserving and stabilising proteins, which can be used for the industrial development of formulations of sanitary, pharmaceutical and cosmetic products that, unlike conventional methods, it takes place in oily phase so that it is more simple and economical, having noted that, at least by the applicant, the existence of any document or invention that discloses a method for preserving and stabilising proteins or similar invention that has technical characteristics similar to those here proposed is unaware.

These and other objects and advantages of the present invention will become apparent from a reading of the attached specification and appended claims.

BRIEF SUMMARY OF THE INVENTION

The present invention is a method which involves the creation of a dispersed system as a means for preservation, stabilization and storage of proteins in an oily phase under normal pressure and temperature conditions. The method of the present invention is based on the incorporation of proteins, such as cell growth factors such as tales epidermal growth factor and fibroblast growth factor, in a medium composed of components, such as grape seed oil and a base including various components which form the oily phase. In particular, the method of the present invention takes advantage of some chemical groups of the oily phase components to promote certain physical-chemical interactions with the residues of proteins that enhance, for longer periods of time, the maintenance of the native molecular structure of proteins. The method is simple, economical and of general application. This method has the potential capacity to replace complex preservation techniques and/or aqueous means commonly used for the preservation of proteins.

In particular, this method for the preservation and stabilizing a proteins includes the steps of forming an anhydrous medium having oily phase components having hydrophilic residues; and dispersing the proteins into the anhydrous medium under ambient pressure and temperature conditions so that the proteins are incorporated into the anhydrous medium so as to maintain an active formation of the proteins. The oily phase components can comprise a base having caprylic/capric triglyceride, a ricinoleic acid, propylene glycol, pentaerythritol tetrakis (3-(3,5-di-tert-butyl-4-hydroxyphenol)) propionate, a tocopherol, and a triisopropanolamine.

This foregoing Section is intended to describe, with particularity, the preferred embodiment of the present invention. It is understood that modifications to this preferred embodiment can be made within the scope of the present claims. As such, this Section should not to be construed, in any way, as limiting of the broad scope of the present invention. The present invention should only be limited by the following claims and their legal equivalents.

DETAILED DESCRIPTION OF THE INVENTION

A medium for dispersing proteins the components of which provides the medium with an oily character has been developed. Thus, the EGF, bFGF and other cell growth factors and/or proteins, as stated, are macromolecules with difficult stabilization, since, currently, the majority of dispersion means have an aqueous character and achieve the stabilization in a short period of time, through the use of dispersed systems, such as emulsions, or expensive and very specific methods that do not ensure a longer life of the protein native structure, such as lyophilisation.

With the present invention, the development of the EGF and other growth factors and/or proteins has been carried out in an oily medium as an intermediate product for use in specific industrial processes, promoting the stability of proteins with respect to the currently existing methods thus achieving a more effective action to the non-denatured proteins in such medium not being denatured the proteins in such medium.

More specifically, according to the method of the present invention, in the formulation the growth factors are surrounded by an anhydrous medium composed of other components that act as adjuvants interacting with the residues of proteins, such components being: grape seed oil, that creates a medium which reduces the electrostatic interactions with the protein residues while maintaining the native conformation of the proteins; base consisting of: caprylic/capric triglyceride; a castor oil; a ricinoleic acid; a propylene glycol; a pentaerythrityl tetrakis (3-(3,5-di-tert-butyl-4-hydroxyphenol)) propionate, a tocopherol, a triisopropanolamine that promotes the interactions by intermolecular forces with the domains of proteins making the structure thermodynamically more stable.

The triisopropanolamine is an amine that is used in a variety of applications and, in particular, as an emulsifier and stabilizer. The triisopropanolamine is also used to neutralize some acidic components. The oil derived from the seed of the Ricinus communis plant and its primary constituent, ricinoleic acid, along with certain of its salts and esters function primarily as skin-conditioning agents, emulsion stabilizers, and surfactants in cosmetics, although other functions also occur. The castor oil and ricinoleic acid can enhance the transdermal penetration of the other chemicals.

Note that the inter-position process of the protein with the oily phase components is carried out with appropriate quality. Such a process is preferably carried out in clean rooms, under laminar flow conditions, where environmental quality controls as well as microbiological controls are guaranteed to ensure the sterility of the product.

In short and succinctly, the present invention proposes the development of a method for the preservation, storage and stabilization ofproteins which contemplates an anhydrous dispersion phase (i.e., in the absence of water) with environmental and microbiological quality, through the application of oily substances having hydrophilic residues that guarantee interactions with the proteins that keep its conformation in the native state, constituting a reproducible, simple and economic method with regard to others methods that require the use of devices, complex methods and qualified personnel.

The foregoing disclosure and description of the invention is illustrative and explanatory thereof. Various changes in the details of the steps of the described method can be made within the scope of the present claims without departing from the true spirit of the invention. The present invention should only be limited by the following claims and their legal equivalents.

BIBLIOGRAPHY

Chang L L, Pikal M J. Mechanisms of Protein Stabilization in the Solid State. J. Pharm. Sci. 98; 2009: 2886-2908.

Chi E Y, Krishnan S, Randolph T W, Carpenter J F. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003; 20: 1325-36.

Krishnamurthy R, Manning M C. The stability factor: importance in formulation development. Curr Pharm Biotechnol. 2002; 3: 361-71.

Schouest J M, Lun T K, Moy R L. Improved texture and appearance of barley produced, synthetic, human-like epidermal growth factor (EGF) serum. J. Drugs Dermatol. 2012; 11 (5): 613-620.

Tang Z, Zhang Z, Zheng Y et al. Cell aging of human diploid fibroblasts is associated with changes in responsiveness to epidermal growth factor and changes in HER-2 expression. Mechanisms of Ageing and Development 1994; 73 (1): 57-67 

I claim:
 1. A method for preserving and stabilizing proteins, the method comprising: forming an anhydrous medium having oily phase components having hydrophilic residues; and dispersing the proteins into the anhydrous medium under ambient pressure and temperature conditions so that the proteins are incorporated into the anhydrous medium so as to maintain an active native formation of the proteins, said oily phase components comprising a base having caprylic/capric triglyceride, a castor oil, a ricinoleic acid, a propylene glycol, pentaerythritol tetrakis (3-(3,5-di-tert-butyl-4-hydroxyphenol)) propionate, a tocopherol, and a triisopropanolamine.
 2. A method for preserving and stabilizing proteins, the method comprising: forming an anhydrous medium having oily phase components having hydrophilic residues; and dispersing the proteins into the anhydrous medium under ambient pressure and temperature conditions so that the proteins are incorporated into the anhydrous medium so as to maintain an active native formation of the proteins, wherein said oily phase components comprise grape seed oil, and a base wherein said base comprises a caprylic or capric triglyceride, a castor oil, a ricinoleic acid, a propylene glycol, pentaerythritol tetrakis (3-(3,5-di-tert-butyl-4-hydroxyphenol)) propionate, a tocopherol, and a triisopropanolamine. 